Electrophoresis is one of the most widely used separation techniques in the biologically-related sciences. Molecular species such as peptides, proteins, and oligonucleotides are separated by causing them to migrate in a buffer solution under the influence of an electric field. This buffer solution normally is used in conjunction with a low to moderate concentration of an appropriate gelling agent such as agarose or polyacrylamide to separate analytes by size and to minimize the occurrence of convective mixing.
Two primary separating mechanisms exist, separations based on differences in the effective charge of the analytes, and separations based on molecular size. The first of these mechanisms is limited in the case of separations of oligonucleotides because in the high molecular weight range the effective charges of these materials become rather similar, making it difficult or impossible to separate them. In the case of proteins, charge and size can be used independently to achieve separations. Separations based on molecular size are generally referred to as molecular sieving and are carried out employing as the separating medium gel matrices having controlled pore sizes. In such separating systems, if the effective charges of the analytes are the same, the separation results from differences in the abilities of the different sized molecular species to penetrate through the gel matrix. Smaller molecules move relatively more quickly than larger ones through a gel of a given pore size. Oligonucleotides and medium to high molecular weight polypeptides and proteins are commonly separated by molecular sieving electrophoresis. In the case of proteinaceous materials, however, it is first necessary to modify the materials to be separated so that they all have the same effective charges. This is commonly done by employing an SDS-PAGE derivatization procedure, such as is discussed in "Gel Electrophoresis of Proteins," B. D. Hames and D. Rickwood, Eds., published by IRL Press, Oxford and Washington, D.C., 1981. The contents of this book are hereby incorporated herein by reference.
Sometimes it is desirable to separate proteinaceous materials under conditions which pose a minimal risk of denaturation. In such cases system additives such as urea and SDS are avoided, and the resulting separations are based on differences in both the molecular sizes and charges
Most electrophoretic separations are today conducted in slabs or open beds. However, such separations are hard to automate or quantitate. Additionally, polyacrylamide gel layers are known to be rather fragile and easily broken. To impart elasticity to such gel layers and to control viscosity, Ogawa has employed water-soluble polymeric additives such as poly(vinyl alcohol), poly(vinyl pyrrolidone), polyacrylamide, polyethylene glycol and polypropylene glycol. See, for example, U.S. Pat. Nos. 4,699,705; 4,657,656; 4,600,641; and 4,582,686. A further problem with flat gel layers for electrophoresis is that they suffer from Joule heating effects under high fields.
Extremely high resolution separations of materials having different effective charges have been achieved by open tubular free-zone electrophoresis and isotachophoresis in narrow capillary tubes. In addition, bulk flow can be driven by electroosmosis to yield very sharp peaks. Such open tubular electrophoresis is not applicable to the separation of medium to high molecular weight oligonucleotides, however, since these materials have very similar effective charges, as indicated above. In addition, open tubular electrophoresis does not provide size selectivity for proteinaceous materials. The questions thus arise whether electrophoresis on gel-containing microcapillaries can be employed to achieve high resolution separations of oligonucleotides, whether non-denaturing gels in such microcapillaries can be used to separate proteinaceous materials, and whether the conventional procedure of SDS-PAGE can be accomplished on such microcapillaries. As demonstrated by the present disclosure, the answers to these questions are yes, although given its potential importance as a separating technique in the biological sciences, surprisingly little attention has been paid to microcapillary gel electrophoresis. Hjerten has published an article in the Journal of Chromatography, 270, 1-6 (1983), entitled "High Performance Electrophoresis: The Electrophoretic Counterpart of High Performance Liquid Chromatography," in which he employs a polyacrylamide gel in tubes having inside dimensions of 50-300 micrometers, and wall thicknesses of 100-200 micrometers. However, this work suffers from limited efficiency and relatively poor performance due in part to the use of relatively wide bore capillaries, relatively low applied fields, and high electrical currents. He has also obtained a patent, U.S. Pat. No. 3,728,145, in which he discloses a method for coating the inner .wall of a large bore tube with a neutral hydrophilic substance such as methyl cellulose or polyacrylamide to reduce electroendosmosis.
In microcapillary gel electrophoresis, resolution between two compounds is influenced by all the factors which affect band sharpness, including sample size, ionic materials in the samples, and the gel concentration. The latter factor is especially important, since if the gel concentration is too high the analytes are totally excluded from the column, while if it is too low no molecular sieving occurs. No single gel concentration is optimal for the resolution of all mixtures of proteinaceous materials or oligonucleotides. It is necessary to select appropriate gel concentrations for particular samples. Other important variables affecting electrophoresis in microcapillaries are the applied field and the electrical current employed.
The current employed in microcapillary electrophoresis is proportional to the square of the tube radius, and the power dissipated is proportional to the square of the current employed at a given voltage. To keep heating effects low therefore requires low currents, which in turn implies the need to use tubes having as small a radius as reasonably possible.
Regarding the effect of the applied field on the resolution attainable in capillary electrophoresis, assuming band broadening is due only to axial diffusion, Giddings in Separation Science, 4, 181-189 (1969) has derived the equation: ##EQU1## where R.sub.s is the resolution achievable between two components of a mixture, .DELTA..mu. is the difference in electrophoretic mobility of two consecutive solutes, E is the applied electric field, t is the time for the electrophoretic analysis, and D is the diffusion coefficient of solutes in the medium (generally taken for proteins to be about 10.sup.-6 cm.sup.2 /sec). As ##EQU2## where L is the tubing length from injection to the point of detection and .mu..sub.ep is the electrophoretic mobility, substituting in the above equation for t yields ##EQU3## An examination of these equations shows that for the case where band broadening is due to axial diffusion, increasing the applied electric field E should increase the resolution in all circumstances. However, it is elementary that increasing the applied potential also increases the current, which in turn increases the heat which must ultimately be dissipated. Thus in the final analysis, for best resolution one must use the highest applied electric fields consistent with manageable thermal effects. It would therefore be very desirable to have gel-containing microcapillary electrophoresis columns which can tolerate the application of high applied electric fields and which do not suffer from thermal effects produced by such fields.